USPTO Patents Application 10714000

Attorney Docket No.: P1746R1P1
Appln. No.: 10/7 14,000
Express Mail No. EV 385 656 535 US

Amendments to the Specification

Please replace the paragraph beginning at page 25, line 19 with the following paragraph:

DHFR, the desired protein and GFP can be expressed from one promoter to improve the
co-expression efficiency. For example, GFP and DHFR can be expressed as a fusion protein, or
an IRES can obviate the need for a second promoter to express GFP. In the constructs shown in
Figure [[9]]i, rows 1 and 2, the exemplary amplifiable selectable gene, DHFR, is fiised to the
GFP gene to form a DHFR-GFP fusion gene. Each of the upstream and downstream coding
sequences (in the first example in Figure [[9]]i, row 1, the upstream coding sequence is DHFR-
GFP fusion gene; in the second example represented in row 2, the upstream coding sequence is
the selected sequence) has its translational stop signal. Translation initiates again for the
downstream coding sequence. These scenarios allow expression of two separate proteins from a
single promoter. It will be understood that the positioning of the promoter/enhancer, translational
stop signal, translational initiation site, transcription termination site and polyA signal, relative to
the various components in each transcription unit, as described here, apply to all the constructs
described below.

Please replace the paragraph beginning at page 27, line 34 with the following paragraph:

The constructs of the invention can also comprise two expression/transcription units, as
shown in Figure [[9]]i, rows 4-9. The two-transcription unit construct depicted in Figure [[9]]i,
row 4, comprises one selected sequence. Rows 5-9 show constructs wherein two selected
sequences can be inserted, one in each transcription unit. Each of the two transcription units will
comprise a promoter and optionally, an enhancer, a transcriptional termination site and polyA
signal sequence. The second transcription unit can use the same or different kind of promoter as
used in first transcription unit. For example, both transcription units can use the SV40 promoter.
One or both of the transcription units can comprise an intron.

Please replace the paragraph beginning at page 28, line 3 with the following paragraph:

Figure [[9]]L row 4, illustrates a construct wherein the first transcription unit contains
DHFR in an intron (the first intron), followed by the selected sequence. The second transcription
unit will comprise the GFP gene. The second transcription unit will preferably comprise an
intron (referred to as the second intron) immediately 5' of the GFP. The three coding sequences
are still physically linked in one vector but are independently transcribed from two promoters.

#189106

2

Attorney Docket No.: P1746R1P1
Appln. No.: 10/714,000
Express Mail No. EV 385 656 535 US

The primary transcript produced from the first transcription unit encodes both DHFR and the
selected sequence but only the DHFR gene is translated into product. Preferably, at least 95% of
the transcripts will have the DHFR gene spliced out and will translate into the desired product. In
the second transcription unit, if the GFP is placed downstream of an intron, both spliced and
unspliced transcripts from this transcription unit will produce GFP.

Please replace the paragraph beginning at page 28, line 19 with the following paragraph:

In yet another embodiment of the preceding construct comprising two transcription units
and two introns, instead of placing the GFP gene within the second intron in the second
transcription unit, an IRES is placed between the second selected sequence and the GFP gene
(Fig. [[9]]i, row 6). Both the second selected sequence and the GFP gene from the second
transcription unit will be translated from the dicistronic message.

Please replace the paragraph beginning at page 28, line 28 with the following paragraph:

In still another variation of the construct comprising two-transcription units and two
introns, the first intron in the first transcription unit is left empty but an IRES is inserted
downstream of the first gene of interest to allow translation of a downstream DHFR-GFP fusion
gene. The second transcription unit will comprise the second intron followed by a second gene of
interest (Fig. [[9]]i, row 8). Optionally, another selectable marker gene (other than the
amplifiable selectable gene and GFP gene), can be placed within the second intron or the intron
can remain without an inserted gene.

#189106

3